Detection of Nucleic Acids Through Amplification of Surrogate Nucleic Acids

ABSTRACT

Methods for detecting and optionally quantitating one or more target nucleic acids are provided, in which a surrogate nucleic acid is captured to each target nucleic acid, amplified, and detected. Compositions, kit, and systems related to the methods are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional utility patent applicationclaiming priority to and benefit of the following prior provisionalpatent application: U.S. Ser. No. 60/735,808, filed Nov. 10, 2005,entitled “DETECTION OF NUCLEIC ACIDS THROUGH AMPLIFICATION OF SURROGATENUCLEIC ACIDS” by Yuling Luo, which is incorporated herein by referencein its entirety for all purposes.

FIELD OF THE INVENTION

The present invention is in the field of nucleic acid detection. Theinvention includes methods for detecting target nucleic acids byspecifically associating surrogate nucleic acids with the target nucleicacids and then amplifying and detecting the surrogate nucleic acids. Theinvention also includes compositions and kits related to the methods.

BACKGROUND OF THE INVENTION

A wide variety of applications in basic biomedical and clinical researchand in molecular medicine require specific detection of one or morenucleic acids (e.g., studies of gene expression). Levels of RNAexpression, for example, have traditionally been measured using Northernblot and nuclease protection assays. However, these approaches aretime-consuming and have limited sensitivity, and the data generated aremore qualitative than quantitative in nature. A multiplex screeningassay for mRNA was recently reported that combines nuclease protectionwith luminescent array detection (Martel et al. (2002) “Multiplexedscreening assay for mRNA combining nuclease protection with luminescentarray detection” Assay Drug Dev Technol. 1:61-71). However, althoughthis assay has the advantage of measuring mRNA transcripts directly fromcell lysates, limited assay sensitivity and reproducibility werereported.

Another exemplary technique, the real-time or quantitative polymerasechain reaction (qPCR), has been gaining widespread use in quantificationof nucleic acid since its development (Higuchi et al. (1993) “KineticPCR analysis: Real-time monitoring of DNA amplification reactions”Biotechnology 11:1026-1030). This is primarily due to qPCR's excellentdetection sensitivity and broad dynamic range, simple homogeneous assayformat, and quantitative capability. However, the quantitativecapability of the qPCR assay for mRNA is significantly compromised bythe pre-analytical steps of RNA isolation and conversion to cDNA, whichresult in significant drops in assay reproducibility and accuracy (see,e.g., Bustin (2000) “Absolute quantification of mRNA using real-timereverse transcription polymerase chain reaction assays” Journal ofMolecular Endocrinology 25:169-193, Bustin (2002) “Quantification ofmRNA using real-time reverse transcription PCR (RT-PCR): Trends andproblems” J Mol Endocrinol 29:23-39, and Bustin and Nolan (2004)“Pitfalls of quantitative real-time reverse-transcription polymerasechain reaction” J Biomol Tech. 15:155-66). There are several reasons whythe pre-analytical steps potentially result in qPCR assay variations.First, isolated RNA can be of variable quality and stability. Second,the efficiency of conversion of RNA to cDNA is dependent on manyfactors, including reverse transcription enzyme efficiency, the presenceof inhibitors in the reverse transcription reaction, template abundance,the presence of background nucleic acids, and different reversetranscription priming methods. Third, extensive degradation andmodification of RNA can occur in samples such as formalin-fixed paraffinembedded tissue, which substantially affect the efficiency of RNAisolation from those samples and the quality of conversion of theisolated RNA to cDNA. Because of the need for RNA isolation and cDNAconversion in the current qPCR format, additional issues arise such asgenomic DNA contamination, 3′ bias, presence of inhibitors in the PCRreaction, interference by other cDNAs within the nucleic acid mixture,amplification efficiency variation among different samples, mispriming,and primer-dimer formation, among others.

Furthermore, the current qPCR format is based on target amplification,and, as a result, target-specific primers and probes need to be designedand validated for every target analyzed. Because the primers and probesare designed using a single gene sequence of limited genetic complexity,yet the PCR is conducted in the presence of a complex cDNA mixture,usually several primer and probe pairs need to be designed and validatedin order to select a primer and probe pair with close to 100%amplification efficiency and no primer-dimer formation. In a multiplexqPCR format, mutual interference of multiple sets of PCR primers andprobes can exacerbate the primer and probe selection problem,substantially increasing the amount of work required for assay designand validation. Also, a qPCR reaction for quantification of a particulartarget nucleic acid usually requires upfront optimization in primer andprobe concentration, in Mg²⁺ and dNTP concentrations, and in hot-startPCR to achieve highest amplification efficiency and to preventmispriming and primer-dimer formation. Finally, assay reproducibility isparticularly problematic when working with very low copy numbers oftarget nucleic acids because of stochastic effects. Particledistribution statistics predict that a greater number of replicates isrequired to differentiate five from 10 copies of a target molecule thanto differentiate 500 from 1000 copies.

Therefore, there is a significant need to develop a qPCR and/or othernucleic acid detection method that eliminates the steps of RNA isolationand reverse transcription. There is also a significant need to develop anucleic acid detection and/or quantification method that does notinvolve amplification of the target nucleic acid. Among other aspects,the present invention provides methods that overcome the above notedlimitations and that permit rapid, simple, and sensitive detectionand/or quantitation of nucleic acids. A complete understanding of theinvention will be obtained upon review of the following.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of detectingtarget nucleic acids through capture and detection of surrogate nucleicacids. Compositions and kits related to the methods are also provided.

A first general class of embodiments provides methods of detecting afirst target nucleic acid. In the methods, a first surrogate nucleicacid and a sample comprising the first target nucleic acid are provided.The first surrogate nucleic acid is physically associated with the firsttarget nucleic acid, to provide captured first surrogate nucleic acid.The captured first surrogate nucleic acid is amplified to provideamplified first surrogate nucleic acid, and the amplified firstsurrogate nucleic acid is detected. Since the amount of amplified firstsurrogate nucleic acid is proportional to the amount of first surrogatenucleic acid captured on the first target nucleic acid, and therefore tothe amount of first target nucleic acid, presence or amount of theamplified first surrogate nucleic acid detected provides an indicationof presence or amount of the first target nucleic acid in the sample.

In one class of embodiments, the first surrogate nucleic acid isassociated with the first target nucleic acid at a molar ratio of about1:1. In another class of embodiments, the first surrogate nucleic acidis associated with the first target nucleic acid at a molar ratio of atleast about 2:1, at least about 3:1, at least about 5:1, at least about10:1, at least about 20:1, or more first surrogate nucleic acid:firsttarget nucleic acid.

In a preferred aspect, a nucleic acid including the first surrogatenucleic acid is physically associated with the first target nucleic acidby hybridization of one or more surrogate capture probes to both nucleicacids. Thus, in one class of embodiments, a first set of one or moresurrogate capture probes is provided. Each of the surrogate captureprobes is capable of hybridizing simultaneously to the first targetnucleic acid and to a nucleic acid comprising the first surrogatenucleic acid. The first set of one or more surrogate capture probes ishybridized to the first target nucleic acid and to the nucleic acidcomprising the first surrogate nucleic acid, whereby the first surrogatenucleic acid is physically associated with the first target nucleicacid. The first set of surrogate capture probes optionally comprises twoor more surrogate capture probes (e.g., three or more, four or more,five or more, or ten or more). Typically, the surrogate capture probeshybridize to nonoverlapping polynucleotide sequences in the first targetnucleic acid.

In one class of embodiments, one or more copies of the nucleic acidcomprising the first surrogate nucleic acid (and therefore of the firstsurrogate nucleic acid itself) are associated with a copy of the firsttarget nucleic acid, the first set of surrogate capture probes comprisesa subset of surrogate capture probes for each of the one or more copiesof the nucleic acid comprising the first surrogate nucleic acid, andeach subset of surrogate capture probes comprises n surrogate captureprobes, where n is at least two. For example, n can be two, three, ormore. In one class of embodiments, hybridizing each subset of nsurrogate capture probes to a copy of the nucleic acid comprising thefirst surrogate nucleic acid associates that copy with the first targetnucleic acid, while hybridization of n−1 of the n surrogate captureprobes to the copy of the nucleic acid comprising the first surrogatenucleic acid does not associate it with the first target nucleic acid.Similarly, in one class of embodiments, hybridizing the first set of oneor more surrogate capture probes to the first target nucleic acid and tothe nucleic acid comprising the first surrogate nucleic acid isperformed at a hybridization temperature which is greater than a meltingtemperature T_(m) of a complex between each individual surrogate captureprobe and the nucleic acid comprising the first surrogate nucleic acid.

The methods optionally include associating the first target nucleic acidwith a solid support, e.g., a multiwell plate or a plurality ofparticles. At any of various steps, materials not associated with thesolid support are optionally separated from the solid support. In oneclass of embodiments, a first set of m target capture probes capable ofhybridizing to the first target nucleic acid, where m is at least one,is provided. The first set of target capture probes is hybridized withthe first target nucleic acid, and the first set of target captureprobes is associated with the solid support, associating the firsttarget nucleic acid with the solid support. As noted, the first set oftarget capture probes includes m target capture probes, where m is atleast one. Preferably, m is at least two. The m target capture probes inthe first set preferably hybridize to nonoverlapping polynucleotidesequences in the first target nucleic acid.

In one class of embodiments, a first support capture probe is bound tothe solid support, and the first set of target capture probes isassociated with the solid support by hybridizing the target captureprobes of the first set with the first support capture probe. Inembodiments in which two or more target capture probes bind to thetarget nucleic acid, binding of a single target capture probe (or lessthan the full set of target capture probes) is optionally insufficientto capture the target nucleic acid to the solid support. Thus, in oneclass of embodiments in which m is at least two, hybridizing the firstset of m target capture probes to the support capture probe captures thefirst target nucleic acid on the solid support, while hybridization ofm−1 of the target capture probes to the support capture probe does notcapture the first target nucleic acid on the solid support. Similarly,in one class of embodiments in which m is at least two, hybridizing thefirst set of target capture probes with the first support capture probeis performed at a hybridization temperature which is greater than amelting temperature T_(m) of a complex between each individual targetcapture probe and the support capture probe.

The captured first surrogate nucleic acid can be amplified byessentially any convenient technique. As just one example, amplifyingthe captured first surrogate nucleic acid to provide amplified firstsurrogate nucleic acid and detecting the amplified first surrogatenucleic acid can comprise performing a quantitative real-time PCRexperiment. The methods are optionally used to determine a relativeamount or an absolute amount of the first target nucleic acid present inthe sample.

The methods can be used to detect target nucleic acids from essentiallyany type of sample. The sample optionally includes a cell lysate, atissue homogenate, an intercellular fluid, a bodily fluid, and/or aconditioned culture medium, and is optionally derived from a tissue(e.g., a formalin-fixed paraffin embedded tissue), a biopsy, and/or atumor. Similarly, the target nucleic acid can be essentially any desirednucleic acid. As just a few examples, the first target nucleic acid canbe derived from one or more of an animal, a human, a plant, a culturedcell, a microorganism, a virus, a bacterium, or a pathogen. The firsttarget nucleic acid can be essentially any type of nucleic acid, e.g., aDNA, an RNA, or an mRNA. Similarly, the first surrogate nucleic acid canbe essentially any type of nucleic acid, e.g., a DNA.

The methods can be conveniently multiplexed for detection of two or moretarget nucleic acids. Thus, in one aspect, the sample including thefirst target nucleic acid also includes a second target nucleic acid,and the methods include providing a second surrogate nucleic acid. Thesecond surrogate nucleic acid is physically associated with the secondtarget nucleic acid, to provide captured second surrogate nucleic acid.The captured second surrogate nucleic acid is amplified to provideamplified second surrogate nucleic acid, and the amplified secondsurrogate nucleic acid is detected. Presence or amount of the amplifiedsecond surrogate nucleic acid detected provides an indication ofpresence or amount of the second target nucleic acid in the sample. Inone class of embodiments, detecting the amplified first surrogatenucleic acid and the amplified second surrogate nucleic acid comprisesphysically separating the amplified first surrogate nucleic acid fromthe amplified second surrogate nucleic acid. The methods optionallyinclude providing, capturing, and amplifying third, fourth, fifth, etc.surrogate nucleic acids as well, such that from two to essentially anydesired number of targets can be detected simultaneously.

Compositions useful in practicing or produced by the methods herein formanother feature of the invention. Thus, another general class ofembodiments provides a composition that includes a first target nucleicacid, a nucleic acid comprising a first surrogate nucleic acid, a firstset of one or more surrogate capture probes, each of which is capable ofhybridizing simultaneously to the first target nucleic acid and to thenucleic acid comprising the first surrogate nucleic acid, and one ormore primers for amplifying the first surrogate nucleic acid.

The composition optionally also includes other reagents for amplifyingthe first surrogate nucleic acid, for example, a nucleic acidpolymerase, nucleoside or deoxynucleoside triphosphates, and the like.It also optionally includes amplified first surrogate nucleic acidand/or one or more reagents for detecting the amplified first surrogatenucleic acid. In one class of embodiments, the composition includes asolid support to which the target and surrogate nucleic acids can becaptured, for example, a multiwell plate or a plurality of particles.

Essentially any desired number of copies of the surrogate nucleic acidcan be associated with each copy of the target nucleic acid. Thus, inone class of embodiments, the nucleic acid comprising the firstsurrogate nucleic acid is hybridized to the first set of surrogatecapture probes, which surrogate capture probes are hybridized to thefirst target nucleic acid, whereby the first surrogate nucleic acid isphysically associated with the first target nucleic acid at a molarratio of about 1:1. In another class of embodiments, the nucleic acidcomprising the first surrogate nucleic acid is hybridized to the firstset of surrogate capture probes, which surrogate capture probes arehybridized to the first target nucleic acid, whereby the first surrogatenucleic acid is physically associated with the first target nucleic acidat a molar ratio of at least about 2:1, at least about 3:1, at leastabout 5:1, or at least about 10:1 first surrogate nucleic acid:firsttarget nucleic acid.

Essentially all of the features described for the methods above apply tothese embodiments as well, as relevant, for example, with respect tonumber and configuration of surrogate capture probes, number andconfiguration of target capture probes, support capture probes, types oftarget and surrogate nucleic acids, and/or the like.

It is worth noting that the composition optionally also includes asecond nucleic acid comprising a second surrogate nucleic acid, a secondset of one or more surrogate capture probes, each of which surrogatecapture probes is capable of hybridizing simultaneously to a secondtarget nucleic acid and to the second nucleic acid comprising the secondsurrogate nucleic acid, the second target nucleic acid, one or moreprimers for amplifying the second surrogate nucleic acid, amplifiedsecond surrogate nucleic acid, and/or one or more reagents for detectingthe amplified second surrogate nucleic acid. Third, fourth, fifth, etc.target and surrogate nucleic acids, sets of surrogate capture probes,and the like are optionally also present in the composition.

Yet another general class of embodiments provides a kit for detecting atleast one target nucleic acid. The kit includes a nucleic acidcomprising a first surrogate nucleic acid, a first set of one or moresurrogate capture probes, each of which is capable of hybridizingsimultaneously to a first target nucleic acid and to the nucleic acidcomprising the first surrogate nucleic acid, a solid support comprisinga first support capture probe bound to the solid support, and a firstset of m target capture probes, where m is at least one, which targetcapture probes are capable of hybridizing simultaneously to the firsttarget nucleic acid and to the first support capture probe, packaged inone or more containers.

The kit optionally also includes one or more primers for amplifying thefirst surrogate nucleic acid, other reagents for amplifying the firstsurrogate nucleic acid (e.g., a nucleic acid polymerase, nucleoside ordeoxynucleoside triphosphates, and the like), one or more reagents fordetecting an amplified first surrogate nucleic acid (e.g., a dye or afluorescently labeled primer or probe), a wash buffer for removingmaterials not specifically captured on the solid support, a lysis bufferfor lysing cells and/or homogenizing tissues, the target and/or thesurrogate nucleic acid at a standard concentration, and/or instructionsfor using the kit to detect and optionally quantitate one or morenucleic acids.

Essentially all of the features described for the methods andcompositions above apply to these embodiments as well, as relevant, forexample, with respect to the number of copies of the surrogate nucleicacid associated with each copy of the target nucleic acid, number andconfiguration of surrogate capture probes, number and configuration oftarget capture probes, support capture probes, types of target andsurrogate nucleic acids, type of solid support, and/or the like.

It is worth noting that the kit optionally also includes a secondnucleic acid comprising a second surrogate nucleic acid, a second set ofone or more surrogate capture probes, each of which surrogate captureprobes is capable of hybridizing simultaneously to a second targetnucleic acid and to the second nucleic acid comprising the secondsurrogate nucleic acid, and a second set of m target capture probes,where m is at least one, which target capture probes are capable ofhybridizing simultaneously to the second target nucleic acid and to thefirst support capture probe. One or more primers for amplifying thesecond surrogate nucleic acid and/or one or more reagents for detectingthe amplified second surrogate nucleic acid can also be included in thekit, as can third, fourth, fifth, etc. target and surrogate nucleicacids, sets of target and surrogate capture probes, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Panels A-E schematically illustrate different schemes forphysically associating one or more copies of a surrogate nucleic acidwith a target nucleic acid.

FIG. 2 Panels A-F depict an exemplary embodiment in which a targetnucleic acid is captured on a solid support and a surrogate nucleic acidis physically associated with the target nucleic acid and thenamplified.

FIG. 3 Panel A schematically illustrates an exemplary setup fordetermining background. Panels B-D schematically illustrate exemplarypotential sources of background.

FIG. 4 Panel A presents a graph illustrating that inclusion of magneticbeads in a qPCR reaction has minimal effect. Panel B presents a graphillustrating detection of an IL-6 RNA target by capture andamplification of a surrogate nucleic acid.

Figures are not necessarily to scale.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not to be imputed to any related or unrelated case, e.g., to anycommonly owned patent or application. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice for testing of the present invention, the preferred materialsand methods are described herein. Accordingly, the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a molecule”includes a plurality of such molecules, and the like.

The term “about” as used herein indicates the value of a given quantityvaries by +/−10% of the value, or optionally +/−5% of the value, or insome embodiments, by +/−1% of the value so described.

The term “nucleic acid” (and the equivalent term “polynucleotide”)encompasses any physical string of monomer units that can becorresponded to a string of nucleotides, including a polymer ofnucleotides (e.g., a typical DNA or RNA polymer), peptide nucleic acids(PNAs), modified oligonucleotides (e.g., oligonucleotides comprisingnucleotides that are not typical to biological RNA or DNA, such as2′-O-methylated oligonucleotides), and the like. The nucleotides of thepolynucleotide can be deoxyribonucleotides, ribonucleotides ornucleotide analogs, can be natural or non-natural, and can beunsubstituted, unmodified, substituted or modified. The nucleotides canbe linked by phosphodiester bonds, or by phosphorothioate linkages,methylphosphonate linkages, boranophosphate linkages, or the like. Thepolynucleotide can additionally comprise non-nucleotide elements such aslabels, quenchers, blocking groups, or the like. The polynucleotide canbe, e.g., single-stranded or double-stranded.

A “target nucleic acid” is a nucleic acid to be detected, for example,via detection of a surrogate nucleic acid specifically associated withthe target nucleic acid.

A “surrogate nucleic acid” is a nucleic acid that can be associated witha target nucleic acid and then amplified, to provide an indication ofwhether the target nucleic acid is present or in what amount the targetnucleic acid is present. Preferably, neither the surrogate nucleic acidnor its complement hybridizes to the target nucleic acid under relevantassay conditions. The surrogate nucleic acid (and its complement)typically has less than 70% or less than 60%, and more typically, lessthan 50% sequence identity with the target nucleic acid. The surrogatenucleic acid is preferably single-stranded.

A “polynucleotide sequence” or “nucleotide sequence” is a polymer ofnucleotides (an oligonucleotide, a DNA, a nucleic acid, etc.) or acharacter string representing a nucleotide polymer, depending oncontext. From any specified polynucleotide sequence, either the givennucleic acid or the complementary polynucleotide sequence (e.g., thecomplementary nucleic acid) can be determined.

Two polynucleotides “hybridize” when they associate to form a stableduplex, e.g., under relevant assay conditions. Nucleic acids hybridizedue to a variety of well characterized physico-chemical forces, such ashydrogen bonding, solvent exclusion, base stacking and the like. Anextensive guide to the hybridization of nucleic acids is found inTijssen (1993) Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays” (Elsevier, N.Y.), as well as in Ausubel, infra.

The “T_(m)” (melting temperature) of a nucleic acid duplex underspecified conditions (e.g., relevant assay conditions) is thetemperature at which half of the base pairs in a population of theduplex are disassociated and half are associated. The T_(m) for aparticular duplex can be calculated and/or measured, e.g., by obtaininga thermal denaturation curve for the duplex (where the T_(m) is thetemperature corresponding to the midpoint in the observed transitionfrom double-stranded to single-stranded form).

The term “complementary” refers to a polynucleotide that forms a stableduplex with its “complement,” e.g., under relevant assay conditions.Typically, two polynucleotide sequences that are complementary to eachother have mismatches at less than about 20% of the bases, at less thanabout 10% of the bases, preferably at less than about 5% of the bases,and more preferably have no mismatches.

A “target capture probe” is a polynucleotide that is capable ofhybridizing to a target nucleic acid. Typically, the target captureprobe is capable of also simultaneously hybridizing to a support captureprobe. The target capture probe typically has a first polynucleotidesequence U-1, which is complementary to the support capture probe, and asecond polynucleotide sequence U-3, which is complementary to apolynucleotide sequence of the target nucleic acid. Sequences U-1 andU-3 are typically not complementary to each other. The target captureprobe is preferably single-stranded.

A “support capture probe” is a polynucleotide that is capable ofhybridizing to at least one target capture probe and that is tightlybound (e.g., covalently or noncovalently, directly or through a linker,e.g., streptavidin-biotin or the like) to a solid support, e.g., amultiwell plate, a slide, a particle, a microsphere, or the like. Thesupport capture probe typically comprises at least one polynucleotidesequence U-2 that is complementary to polynucleotide sequence U-1 of atleast one target capture probe. The support capture probe is preferablysingle-stranded.

A “surrogate capture probe” is a polynucleotide that is capable ofhybridizing to a target nucleic acid and to a nucleic acid including asurrogate nucleic acid. The surrogate capture probe typically has afirst polynucleotide sequence U-4, which is complementary to apolynucleotide sequence of the target nucleic acid, and a secondpolynucleotide sequence U-5, which is complementary to the nucleic acidincluding the surrogate nucleic acid. Sequences U-4 and U-5 aretypically not complementary to each other. The surrogate capture probeis preferably single-stranded.

A nucleic acid is “amplified” when one or more additional nucleic acidmolecules having a nucleotide sequence corresponding to that of thenucleic acid and/or its complement are produced. For example, a nucleicacid can be amplified in a template-dependent reaction in which a primeranneals to the nucleic acid and is extended by a nucleic acid polymeraseto produce a copy of the nucleic acid's complement; optionally, anotherprimer anneals to the complement and is extended to produce a copy ofthe nucleic acid. A single-stranded DNA molecule can be amplified, forexample, by production of a complementary DNA strand or by production ofa complementary RNA strand.

A first polynucleotide that is “capable of hybridizing” (or “configuredto hybridize”) to a second polynucleotide comprises a firstpolynucleotide sequence that is complementary to a second polynucleotidesequence in the second polynucleotide.

A “primer” is a nucleic acid that contains a sequence complementary to aregion of a template nucleic acid strand and that primes the synthesisof a strand complementary to the template (or a portion thereof).Primers are typically, but need not be, relatively short, chemicallysynthesized oligonucleotides (typically, oligodeoxynucleotides). Inorder to be extendable by a standard polymerase, a primer typically hasa free 3′ hydroxyl group.

A “label” is a moiety that facilitates detection of a molecule. Commonlabels in the context of the present invention include fluorescent,luminescent, light-scattering, and/or colorimetric labels. Suitablelabels include enzymes and fluorescent moieties, as well asradionuclides, substrates, cofactors, inhibitors, chemiluminescentmoieties, magnetic particles, and the like. Patents teaching the use ofsuch labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many labels arecommercially available and can be used in the context of the invention.

A “microsphere” is a small spherical, or roughly spherical, particle. Amicrosphere typically has a diameter less than about 1000 micrometers(e.g., less than about 100 micrometers, optionally less than about 10micrometers).

A “microorganism” is an organism of microscopic or submicroscopic size.Examples include, but are not limited to, bacteria, fungi, yeast,protozoans, microscopic algae (e.g., unicellular algae), viruses (whichare typically included in this category although they are incapable ofgrowth and reproduction outside of host cells), subviral agents,viroids, and mycoplasma.

A variety of additional terms are defined or otherwise characterizedherein.

DETAILED DESCRIPTION

In one aspect, the invention provides methods for detecting andoptionally quantitating a target nucleic acid. A surrogate nucleic acidis specifically associated with the target nucleic acid, and thesurrogate nucleic acid is amplified and detected. The target nucleicacid is optionally associated with a solid support and need not bepurified prior to association with the solid support and/or thesurrogate nucleic acid. The methods of the invention can thus avoidproblems associated with RNA isolation, reverse transcription, and thelike. In addition, multiple copies of the surrogate nucleic acid areoptionally associated with each copy of the target nucleic acid,improving sensitivity and precision when a low copy number target is tobe detected. Compositions, kits, and systems related to the methods arealso features of the invention.

Methods

One general class of embodiments provides methods of detecting a firsttarget nucleic acid. In the methods, a first surrogate nucleic acid anda sample comprising the first target nucleic acid are provided. Thefirst surrogate nucleic acid is physically associated with (e.g.,indirectly bound or hybridized to) the first target nucleic acid, toprovide captured first surrogate nucleic acid. The captured firstsurrogate nucleic acid is amplified to provide amplified first surrogatenucleic acid, and the amplified first surrogate nucleic acid isdetected. Since the amount of amplified first surrogate nucleic acid isproportional to the amount of first surrogate nucleic acid captured onthe first target nucleic acid, and therefore to the amount of firsttarget nucleic acid, presence or amount of the amplified first surrogatenucleic acid detected provides an indication of presence or amount ofthe first target nucleic acid in the sample.

A single copy of the surrogate nucleic acid can be associated with eachcopy of the target nucleic acid. Thus, in one class of embodiments, thefirst surrogate nucleic acid is associated with the first target nucleicacid at a molar ratio of about 1:1. See, e.g., FIG. 1 Panels A, C, andE. Alternatively, however, multiple copies of the surrogate nucleic acidcan be associated with each copy of the target nucleic acid, e.g., fromtwo copies to essentially any desired preselected number of copies.Thus, in one class of embodiments, the first surrogate nucleic acid isassociated with the first target nucleic acid at a molar ratio of atleast about 2:1, about 3:1, about 5:1, about 10:1, about 20:1, or morefirst surrogate nucleic acid:first target nucleic acid. See, e.g., FIG.1 Panels B and D.

In a preferred aspect, a nucleic acid including the first surrogatenucleic acid is physically associated with the first target nucleic acidby hybridization of one or more surrogate capture probes to both nucleicacids. Thus, in one class of embodiments, a first set of one or moresurrogate capture probes is provided. Each of the surrogate captureprobes is capable of hybridizing simultaneously to the first targetnucleic acid and to a nucleic acid comprising the first surrogatenucleic acid. The first set of one or more surrogate capture probes ishybridized to the first target nucleic acid and to the nucleic acidcomprising the first surrogate nucleic acid, whereby the first surrogatenucleic acid is physically associated with the first target nucleicacid. Hybridization of the surrogate capture probe(s) to the nucleicacids can occur simultaneously or sequentially, in either order. Thefirst set of surrogate capture probes optionally comprises two or moresurrogate capture probes (e.g., three or more, four or more, five ormore, or ten or more). Typically, the surrogate capture probes hybridizeto nonoverlapping polynucleotide sequences in the first target nucleicacid (see, e.g., FIG. 1 Panels B-D, which depict three exemplary bindingarrangements of surrogate capture probes 103 to target nucleic acid 101and a nucleic acid comprising surrogate nucleic acid 102). Thenonoverlapping polynucleotide sequences are optionally, but need not be,contiguous.

The nucleic acid comprising the surrogate nucleic acid optionallyconsists of the surrogate nucleic acid. In such embodiments, thesurrogate capture probe(s) hybridize to nucleotide sequences within thesurrogate nucleic acid. Typically, however, the nucleic acid includespolynucleotide sequence U-6 in addition to the sequence of the surrogatenucleic acid, and the surrogate capture probe(s) are complementary toU-6 or subsequences within U-6. In one class of embodiments, the nucleicacid comprising the surrogate nucleic acid includes one copy of thesurrogate nucleic acid, while in other embodiments, the nucleic acidcomprising the surrogate nucleic acid includes two or more copies of thesurrogate nucleic acid (e.g., two, three, five, 10, 20, or more copies).Thus, in embodiments in which multiple copies of the surrogate nucleicacid are associated with or captured to each copy of the target nucleicacid, more than one copy of the nucleic acid comprising the surrogatenucleic acid can be associated with the each copy of the target nucleicacid and/or each copy of the nucleic acid comprising the surrogatenucleic acid can include more than one copy of the surrogate nucleicacid.

Each copy of the nucleic acid comprising the surrogate nucleic acid isoptionally associated with the target nucleic acid through hybridizationof a single surrogate capture probe (see, e.g., FIG. 1 Panels A and B).Alternatively, each copy of the nucleic acid comprising the surrogatenucleic acid can be associated with the target nucleic acid throughhybridization of two or more surrogate capture probes. Thus, in oneclass of embodiments, one or more copies of the nucleic acid comprisingthe first surrogate nucleic acid are associated with a copy of the firsttarget nucleic acid, the first set of surrogate capture probes comprisesa subset of surrogate capture probes for each of the one or more copiesof the nucleic acid comprising the first surrogate nucleic acid, andeach subset of surrogate capture probes comprises n surrogate captureprobes, where n is at least two. For example, n can be two, three, ormore. See, e.g., FIG. 1 Panels C and D. The surrogate capture probeswithin a given subset typically hybridize to nonoverlappingpolynucleotide sequences in the nucleic acid comprising the firstsurrogate nucleic acid, as well as to nonoverlapping polynucleotidesequences in the first target nucleic acid. n is typically, but notnecessarily, the same from subset to subset.

In embodiments in which two or more surrogate capture probes are used tocapture each copy of the nucleic acid comprising the surrogate nucleicacid, binding of a single surrogate capture probe (or of less than thefull subset of surrogate capture probes) is optionally too weak tostably capture the nucleic acid comprising the surrogate nucleic acid tothe target nucleic acid. Thus, in one class of embodiments, hybridizingeach subset of n surrogate capture probes to a copy of the nucleic acidcomprising the first surrogate nucleic acid associates that copy withthe first target nucleic acid, while hybridization of n−1 of the nsurrogate capture probes to the copy of the nucleic acid comprising thefirst surrogate nucleic acid does not associate it with the first targetnucleic acid. Similarly, in one class of embodiments, hybridizing thefirst set of one or more surrogate capture probes to the first targetnucleic acid and to the nucleic acid comprising the first surrogatenucleic acid is performed at a hybridization temperature which isgreater than a melting temperature T_(m) of a complex between eachindividual surrogate capture probe and the nucleic acid comprising thefirst surrogate nucleic acid. For example, the hybridization temperaturecan be about 5° C. or more, about 7° C. or more, about 10° C. or more,about 15° C. or more, or about 20° C. or more greater than the T_(m). Inthese embodiments, the T_(m) of the complex between each individualsurrogate capture probe and the first target nucleic acid is preferablysignificantly greater than the hybridization temperature such that eachsurrogate capture probe stably hybridizes to the target nucleic acid atthe hybridization temperature. In alternative exemplary embodiments,binding of each individual surrogate capture probe to the first targetnucleic acid is weak, while hybridization of each surrogate captureprobe to the nucleic acid comprising the first surrogate nucleic acid isstable at the hybridization temperature.

It will be evident that a number of configurations for the surrogatecapture probes are possible. For example, as illustrated in FIG. 1Panels A-D, the 5′ end of each surrogate capture probe can hybridize tothe target nucleic acid while the 3′ end is complementary to the nucleicacid comprising the surrogate nucleic acid (or vice versa). As anotherexample, one surrogate capture probe can have its 5′ end complementaryto the target nucleic acid and its 3′ end complementary to the nucleicacid comprising the surrogate nucleic acid, while another surrogatecapture probe has its 3′ end complementary to the target nucleic acidand its 5′ end complementary to the nucleic acid comprising thesurrogate nucleic acid, resulting in a cruciform arrangement. In yetanother example, one oligonucleotide (106) hybridizes to the targetnucleic acid and to another oligonucleotide (107) which in turnhybridizes to the nucleic acid comprising the surrogate nucleic acid(see FIG. 1 Panel E).

In one aspect, the target nucleic acid, and thus its associatedsurrogate nucleic acid, is captured on a solid support. Thus, in oneclass of embodiments, the methods include associating the first targetnucleic acid with a solid support. Association of the target nucleicacid with the solid support and association of the surrogate nucleicacid with the target nucleic acid can occur simultaneously orsequentially, in either order.

Capture of the target nucleic acid to the solid support optionallyinvolves hybridization of the target nucleic acid to target captureprobes associated with the solid support. Thus, in one class ofembodiments, a first set of m target capture probes capable ofhybridizing to the first target nucleic acid, where m is at least one,is provided. The first set of target capture probes is hybridized withthe first target nucleic acid, and the first set of target captureprobes is associated with the solid support, associating the firsttarget nucleic acid with the solid support. Hybridization of the targetcapture probes with the target nucleic acid and association of thetarget capture probes with the solid support can occur simultaneously orsequentially, in either order.

As noted, the first set of target capture probes includes m targetcapture probes, where m is at least one. Preferably, m is at least twoor at least three, and m can be at least four or at least five or more.Typically, but not necessarily, m is at most ten. For example, m can bebetween three and ten, e.g., between five and ten or between five andseven, inclusive. Use of fewer target capture probes can beadvantageous, for example, in embodiments in which the target nucleicacid is to be specifically captured from samples including other nucleicacids with sequences very similar to that of the target nucleic acid. Inother embodiments (e.g., embodiments in which capture of as much of thetarget nucleic acid as possible is desired), however, m can be more than10, e.g., between 20 and 50. The m target capture probes in the firstset preferably hybridize to nonoverlapping polynucleotide sequences inthe first target nucleic acid. The nonoverlapping polynucleotidesequences can, but need not be, consecutive within the target nucleicacid.

The target capture probes are optionally bound directly to the solidsupport (covalently or noncovalently), or they can be indirectlyassociated with the solid support. In one class of embodiments, a firstsupport capture probe is bound to the solid support, and the first setof target capture probes is associated with the solid support byhybridizing the target capture probes of the first set with the firstsupport capture probe.

In embodiments in which two or more target capture probes bind to thetarget nucleic acid, binding of a single target capture probe (or lessthan the full set of target capture probes) is optionally insufficientto capture the target nucleic acid to the solid support. Thus, in oneclass of embodiments in which m is at least two, hybridizing the firstset of m target capture probes to the support capture probe captures thefirst target nucleic acid on the solid support, while hybridization ofm−1 of the target capture probes to the support capture probe does notcapture the first target nucleic acid on the solid support. Similarly,in one class of embodiments in which m is at least two, hybridizing thefirst set of target capture probes with the first support capture probeis performed at a hybridization temperature which is greater than amelting temperature T_(m) of a complex between each individual targetcapture probe and the support capture probe. For example, thehybridization temperature can be about 5° C. or more, about 7° C. ormore, about 10° C. or more, about 15° C. or more, or about 20° C. ormore greater than the T_(m). In these embodiments, the T_(m) of thecomplex between each individual target capture probe and the firsttarget nucleic acid is preferably significantly greater than thehybridization temperature such that each target capture probe stablyhybridizes to the target nucleic acid at the hybridization temperature.In alternative exemplary embodiments, binding of each individual targetcapture probe to the first target nucleic acid is weak, whilehybridization of each target capture probe to the first support captureprobe is stable at the hybridization temperature.

In embodiments in which a support capture probe is employed, the targetcapture probe typically includes a polynucleotide sequence U-1 that iscomplementary to a polynucleotide sequence U-2 in the support captureprobe. Sequences U-1 and U-2 can be of essentially any convenientlength, depending, e.g., on the G-C base content of U-1 and U-2, on thehybridization temperature, and on whether hybridization between anindividual target capture probe and support capture probe is desired tobe strong or weak. In one aspect, U-1 and U-2 are 20 nucleotides or lessin length. In one class of embodiments, U-1 and U-2 are between 9 and 17nucleotides in length (inclusive), preferably between 12 and 15nucleotides (inclusive). For example, U-1 and U-2 can be 14, 15, 16, or17 nucleotides in length, or they can be between 9 and 13 nucleotides inlength (e.g., for lower hybridization temperatures, e.g., hybridizationat room temperature).

The support capture probe can include polynucleotide sequence inaddition to U-2, or U-2 can comprise the entire polynucleotide sequenceof the support capture probe. For example, each support capture probeoptionally includes a linker sequence between the site of attachment ofthe support capture probe to the solid support and sequence U-2 (e.g., alinker sequence containing 8 Ts, as just one possible example).

It will be evident that the number of target capture probes required forstable capture of a target nucleic acid depends, in part, on the amountof overlap between the target capture probes and the support captureprobe (i.e., the length of U-1 and U-2). For example, if m is 5-7 for a14 nucleotide overlap, m could be 3-5 for a 15 nucleotide overlap or 2-3for a 16 nucleotide overlap.

Stable capture of target nucleic acids, e.g., while minimizing captureof extraneous nucleic acids (e.g., those to which m−1 or fewer of thetarget capture probes bind) can be achieved, for example, by balancing m(the number of target capture probes), the amount of overlap between thetarget capture probes and the support capture probe (the length of U-1and U-2), and/or the stringency of the conditions under which the targetcapture probes, the nucleic acids, and the support capture probes arehybridized.

Appropriate combinations of m, amount of complementarity between thetarget capture probes and the support capture probe, and stringency ofhybridization can, for example, be determined experimentally by one ofskill in the art. For example, a particular value of m and a particularset of hybridization conditions can be selected, while the number ofnucleotides of complementarity between the target capture probes and thesupport capture probe is varied until hybridization of the m targetcapture probes to a nucleic acid captures the nucleic acid whilehybridization of a single target capture probe (or optionally of m−1target capture probes) does not efficiently capture the nucleic acid.Similarly, m, amount of complementarity, and stringency of hybridizationcan be selected such that the desired nucleic acid of interest iscaptured while other nucleic acids present in the sample are notefficiently captured. Stringency can be controlled, for example, bycontrolling the formamide concentration, chaotropic salt concentration,salt concentration, pH, organic solvent content, and/or hybridizationtemperature.

As noted, the T_(m) of any nucleic acid duplex can be directly measured,using techniques well known in the art. For example, a thermaldenaturation curve can be obtained for the duplex, the midpoint of whichcorresponds to the T_(m). It will be evident that such denaturationcurves can be obtained under conditions having essentially any relevantpH, salt concentration, solvent content, and/or the like.

The T_(m) for a particular duplex (e.g., an approximate T_(m)) can alsobe calculated. For example, the T_(m) for an oligonucleotide-targetduplex can be estimated using the following algorithm, whichincorporates nearest neighbor thermodynamic parameters: T_(m)(Kelvin)=ΔH°/(ΔS°+R lnC_(t)), where the changes in standard enthalpy(ΔH°) and entropy (ΔS°) are calculated from nearest neighborthermodynamic parameters (see, e.g., SantaLucia (1998) “A unified viewof polymer, dumbbell, and oligonucleotide DNA nearest-neighborthermodynamics” Proc. Natl. Acad. Sci. USA 95:1460-1465, Sugimoto et al.(1996) “Improved thermodynamic parameters and helix initiation factor topredict stability of DNA duplexes” Nucleic Acids Research 24: 4501-4505,Sugimoto et al. (1995) “Thermodynamic parameters to predict stability ofRNA/DNA hybrid duplexes” Biochemistry 34:11211-11216, and et al. (1998)“Thermodynamic parameters for an expanded nearest-neighbor model forformation of RNA duplexes with Watson-Crick base pairs” Biochemistry 37:14719-14735), R is the ideal gas constant (1.987 cal·K⁻¹mole⁻¹), andC_(t) is the molar concentration of the oligonucleotide. The calculatedT_(m) is optionally corrected for salt concentration, e.g., Na⁺concentration, using the formula 1/T_(m)(Na⁺)=1/T_(m)(1M)+(4.29f(G·C)−3.95)×10⁻⁵ ln[Na⁺]+9.40×10⁻⁶ ln²[Na⁺]. See, e.g., Owczarzy et al.(2004) “Effects of sodium ions on DNA duplex oligomers: Improvedpredictions of melting temperatures” Biochemistry 43:3537-3554 forfurther details. A web calculator for estimating T_(m) using the abovealgorithms is available on the internet at scitools (dot) idtdna (dot)com/analyzer/oligocalc (dot) asp. Other algorithms for calculating T_(m)are known in the art and are optionally applied to the presentinvention.

It will be evident that similar considerations apply to design ofsurrogate capture probes, above; for example, with respect to number ofsurrogate capture probes per subset (n), number of nucleotides ofcomplementarity between the surrogate capture probe and the nucleic acidcomprising the surrogate nucleic acid (length of U-5), and/or thestringency of the conditions under which the surrogate capture probes,the nucleic acid comprising the surrogate nucleic acid, and the targetnucleic acid are hybridized.

The target capture probes and surrogate capture probes are preferablycomplementary to physically distinct, nonoverlapping sequences in thetarget nucleic acid, which can be, but are not necessarily, contiguous.The T_(m)s of the individual target capture probe-target nucleic acidand surrogate capture probe-target nucleic acid complexes are preferablygreater than the hybridization temperature, e.g., by 5° C. or 10° C. orpreferably by 15° C. or more, such that these complexes are stable atthe hybridization temperature. Sequence U-3 for each target captureprobe is typically (but not necessarily) about 17-35 nucleotides inlength, with about 30-70% GC content. Similarly, sequence U-4 for eachsurrogate capture probe is typically (but not necessarily) about 17-35nucleotides in length, e.g., about 20-30 nucleotides.

Potential target capture probe sequences (e.g., potential sequences U-3)are optionally examined for possible interactions with non-correspondingnucleic acids of interest, repetitive sequences (such as polyC or polyT,for example), any primers and/or detection probes used to amplify and/ordetect the surrogate nucleic acid, and/or any relevant genomicsequences, for example; sequences expected to cross-hybridize withundesired nucleic acids are typically not selected for use in the targetcapture probes. Examination can be, e.g., visual (e.g., visualexamination for complementarity), computational (e.g., computation andcomparison of percent sequence identity and/or binding free energies;for example, sequence comparisons can be performed using BLAST softwarepublicly available through the National Center for BiotechnologyInformation on the world wide web at www (dot) ncbi (dot) nlm (dot) nih(dot) gov), and/or experimental (e.g., cross-hybridization experiments).Potential surrogate, support capture probe, and surrogate capture probesequences are preferably similarly examined, to ensure that undesirablecross-hybridization is not expected to occur.

A support capture probe, target capture probe, surrogate capture probe,and/or nucleic acid including a surrogate nucleic acid optionallycomprises at least one non-natural nucleotide. For example, a supportcapture probe and the corresponding target capture probe optionallycomprise, at complementary positions, at least one pair of non-naturalnucleotides that base pair with each other but that do not Watson-Crickbase pair with the bases typical to biological DNA or RNA (i.e., A, C,G, T, or U). Examples of nonnatural nucleotides include, but are notlimited to, Locked NucleicAcid™ nucleotides (available from Exiqon A/S,on the world wide web at www (dot) exiqon (dot) com; see, e.g.,SantaLucia Jr. (1998) Proc Natl Acad Sci 95:1460-1465) and isoG, isoC,and other nucleotides used in the AEGIS system (Artificially ExpandedGenetic Information System, available from EraGen Biosciences, on theworld wide web at www (dot) eragen (dot) com; see, e.g., U.S. Pat. No.6,001,983, U.S. Pat. No. 6,037,120, and U.S. Pat. No. 6,140,496). Use ofsuch non-natural base pairs (e.g., isoG-isoC base pairs) in the supportcapture probes and target capture probes (or the surrogate captureprobes and nucleic acids including surrogate nucleic acids) can, forexample, decrease cross hybridization, or it can permit use of shorterprobes when the non-natural base pairs have higher binding affinitiesthan do natural base pairs.

The solid support can be essentially any suitable support, including anyof a variety of materials, configurations, and the like. For example,the solid support can comprise a multiwell plate or a plurality ofparticles (e.g., microspheres). Supports are discussed in greater detailin the section entitled “Solid supports” below.

At any of various steps, materials not associated with the solid supportare optionally separated from the solid support. For example, after thetarget capture probes, target nucleic acid, and support-bound supportcapture probes are hybridized, the solid support is optionally washed toremove any unbound target capture probes and non-target nucleic acids.Similarly, the support can be washed after association of the surrogatenucleic acid with the target nucleic acid, to remove any unboundsurrogate nucleic acid before amplification and detection of thesurrogate.

The captured first surrogate nucleic acid can be amplified byessentially any convenient technique, depending, e.g., on the nature ofthe first surrogate nucleic acid (e.g., RNA or DNA), the technique to beused for detecting the amplified first surrogate nucleic acid, and/orthe like. The first surrogate nucleic acid is preferably a DNA but canbe an RNA or essentially any other form of nucleic acid. Similarly, theamplified first surrogate nucleic acid is preferably a DNA but can be anRNA or any other form of nucleic acid, and it can be single-stranded ordouble-stranded. One or more cycles of amplification can be performed,depending for example on the degree of amplification desired.

A wide variety of techniques for amplifying and detecting nucleic acidsare known in the art and can be adapted to the practice of the presentinvention. Oligonucleotide primers, a nucleic acid polymerase,nucleoside or deoxynucleoside triphosphates, cofactors, aqueous bufferedsalt solutions, and the like are provided as appropriate for theselected technique, as is well-known in the art. As just a few examples,an RNA first surrogate nucleic acid can be amplified and the resultingdouble-stranded DNA amplified first surrogate nucleic acid detectedusing a reverse transcription-polymerase chain reaction (PCR) technique,e.g., a real-time reverse transcription-PCR technique, or a DNA firstsurrogate nucleic acid can be amplified by T7 polymerase, T3 polymerase,SP6 polymerase, strand displacement amplification, multiple-displacementamplification, or by rolling circle amplification (see, e.g., Hatch etal. (1999) “Rolling circle amplification of DNA immobilized on solidsurfaces and its application to multiplex mutation detection” GenetAnal. 15:35-40; Baner et al. (1998) “Signal amplification of padlockprobes by rolling circle replication” Nucleic Acids Res. 26:5073-8;Nallur et al. (2001) “Signal amplification by rolling circleamplification on DNA microarrays” Nucleic Acids Res. 29:E118; andDemidov and Broude, eds. (2004) DNA Amplification: Current Technologiesand Applications, Horizon Bioscience.)

In one class of embodiments, amplifying the captured first surrogatenucleic acid involves one or more PCR cycles. In PCR, a pair of primersflanking the region to be amplified is typically provided.Template-dependent extension of the primers is catalyzed by a DNApolymerase, in the presence of deoxyribonucleoside triphosphates(typically dATP, dCTP, dGTP, and dTTP, although these can be replacedand/or supplemented with other dNTPs, e.g., a dNTP comprising a baseanalog that Watson-Crick base pairs like one of the conventional bases,e.g., uracil, inosine, or 7-deazaguanine), an aqueous buffer, andappropriate salts and metal cations (e.g., Mg²⁺). The PCR processtypically involves cycles of three steps: denaturation (e.g., ofdouble-stranded template and/or extension product), annealing (e.g., ofone or more primers to template), and extension (e.g., of one or moreprimers to form double-stranded extension products). The PCR process caninstead, e.g., involve cycles of two steps: denaturation (e.g., ofdouble-stranded template and/or extension product) andannealing/extension (e.g., of one or more primers to template and of oneor more primers to form double-stranded extension products). The cyclesare typically thermal cycles; for example, cycles of denaturation attemperatures greater than about 90° C., annealing at 50-75° C., andextension at 60-78° C. A thermostable enzyme is thus preferred. Suchenzymes (including, e.g., Thermus aquaticus Taq DNA polymerase),appropriate buffers, etc. are widely commercially available, e.g., fromClontech (on the world wide web at www (dot) clontech (dot) com),Invitrogen (at www (dot) invitrogen (dot) com), Sigma-Aldrich (at www(dot) sigma-aldrich (dot) com), and New England Biolabs (at www (dot)neb (dot) com). PCR techniques have been extremely well described inboth the patent and the scientific literature, and any of a variety ofsuch techniques can be employed, including, e.g., asymmetric PCR.

In one class of embodiments, amplifying the captured first surrogatenucleic acid to provide amplified first surrogate nucleic acid anddetecting the amplified first surrogate nucleic acid comprisesperforming a quantitative real-time PCR experiment. In real-time PCR,product formation is monitored in real time, for example, at apreselected point in each cycle. In real-time quantitative PCR withfluorescent detection of product, for example, a fluorescence thresholdabove background is typically assigned, and the time point at which eachreaction's amplification plot reaches that threshold (defined as thethreshold cycle number or Ct) is determined. The Ct value can be used tocalculate the quantity of template initially present in each reaction.(Under a standard set of conditions, a lower or higher starting templateconcentration produces a higher or lower, respectively, Ct value.) Themethods are optionally used to determine a relative amount or anabsolute amount of the first target nucleic acid present in the sample.

Real-time PCR techniques have been well described. See, e.g., thereferences noted above and Stephen A. Bustin, ed. (2004) A-Z ofQuantitative PCR, International University Line, Edwards et al., eds.(2004) Real-Time PCR: An Essential Guide, Horizon Bioscience, and Klein(2002) “Quantification using real-time PCR technology: Applications andlimitations” Trends in Molecular Medicine 8:257-260. In addition,automated thermal cyclers, including integrated systems for real timedetection of product, are commercially available, e.g., the ABI Prism®7700 sequence detection system from Applied Biosystems (on the worldwide web at www (dot) appliedbiosystems (dot) com), the iCycler iQ®real-time PCR detection system from Bio-Rad (www (dot) biorad (dot)com), or the DNA Engine Opticon® continuous fluorescence detectionsystem from MJ Research, Inc. (www (dot) mjr (dot) com).

The amplified first surrogate nucleic acid can be detected by any of avariety of techniques well known in the art. Detection optionallyinvolves physical separation of the amplified surrogate nucleic acidfrom any other products of the amplification reaction, e.g., byelectrophoresis, use of a fluorescent nucleic acid-binding dye, orbinding of a labeled sequence-specific oligonucleotide probe, forexample. In embodiments in which a real-time PCR technique is employed,the amplified surrogate nucleic acid can be detected using, for example,a fluorescent dye such as SYBR® green, ethidium bromide, or YO-PRO-1, orone or more fluorescently labeled oligonucleotide probes and/or primers,e.g., TagMan® primers, scorpion primers, HybProbes, Amplifluor®(sunrise) hairpin primers, molecular beacons, or Invader®oligonucleotides. See, e.g., Mackay et al. (2002) “Real-time PCR invirology” Nucleic Acids Res. 30:1292-1305, Poddar (2000) “Symmetric vs.asymmetric PCR and molecular beacon probe in the detection of a targetgene of adenovirus” Molecular and Cellular Probes 14: 25-32, Nazarenkoet al. (1997) “A closed tube format for amplification and detection ofDNA based on energy transfer” Nucl. Acids Res. 25:2516-2521, Marras etal. (1999) “Multiplex detection of single-nucleotide variation usingmolecular beacons” Genet. Anal. Biomol. Eng. 14:151-156, Mhlanga et al.(2001) “Using molecular beacons to detect single-nucleotidepolymorphisms with real-time PCR” Methods 25:463-471, Tyagi and Kramer(1996) “Molecular beacons: probes that fluoresce upon hybridization”Nature Biotechnology 14:303-308; Blok and Kramer (1997) “Amplifiablehybridization probes containing a molecular switch” Mol Cell Probes11:187-194, Hsu et al. (2001) “Genotyping single-nucleotidepolymorphisms by the Invader assay with dual-color fluorescencepolarization detection” Clinical Chemistry 47:1373-1377, U.S. Pat. No.5,691,146 (Nov. 25, 1997) to Mayrand entitled “Methods for combined PCRamplification and hybridization probing using doubly labeled fluorescentprobes”, U.S. Pat. No. 6,277,607 (Aug. 21, 2001) to Tyagi et al.entitled “High specificity primers, amplification methods and kits”,U.S. Pat. No. 5,866,336 (Feb. 2, 1999) to Nazarenko et al. entitled“Nucleic acid amplification oligonucleotides with molecular energytransfer labels and methods based thereon”, and references therein,among many other references.

An exemplary embodiment is schematically illustrated in FIG. 2. Panel Aillustrates solid support 201 to which is bound support capture probe202. Support capture probe 202 includes polynucleotide sequence U-2(206). First set 203 of target capture probes is also illustrated. Eachtarget capture probe includes sequences U-1 (204, complementary to thesupport capture probe's sequence U-2) and U-3 (205, complementary to asequence in first target nucleic acid 210). Nucleic acid 211 includessurrogate nucleic acid 212 and sequence U-6 213. (As illustrated,nucleic acid 211 includes a single copy of the surrogate nucleic acid;in other embodiments, however, nucleic acid 211 optionally includes twoor more copies of the surrogate nucleic acid sequence, for even greateramplification of the signal.) As depicted, first set 220 of surrogatecapture probes includes four surrogate capture probes, one subset of twocomplementary to a first copy of nucleic acid 211 and another subset oftwo complementary to a second copy of nucleic acid 211 (which can haveidentical or distinct sequences U-6). Each of the exemplary surrogatecapture probes includes polynucleotide sequence U-4 (221, complementaryto a sequence in first target nucleic acid 210) at its 5′ end, spacersequence 222 (e.g., five Ts), and sequence U-5 (223, complementary to asequence in region 213 of nucleic acid 211) at its 3′ end.

The target capture probes are hybridized to the support capture probeand to the target nucleic acid, and the surrogate capture probes arehybridized to the target nucleic acid and the nucleic acid including thesurrogate nucleic acid, simultaneously or sequentially (Panel B).Optional blocking probe 230 is hybridized to a region of target nucleicacid 210 not occupied by the target or support capture probes. Materialsnot captured on the solid support (e.g., non-target nucleic acid 240)are optionally separated from the support by washing.

Panels C-F schematically illustrate amplification of surrogate nucleicacid 212. The surrogate nucleic acid is optionally still associated withthe solid support, or it is optionally removed from the solid supportprior to amplification. Primer 250 is provided and annealed to surrogatenucleic acid 212 (Panel C). The primer is extended to produce complement214 of surrogate nucleic acid 212 (Panel D). In embodiments in whichonly one cycle of amplification is performed and/or in which only asingle primer is provided, complement 214 corresponds to the amplifiedfirst surrogate nucleic acid. Optionally, however, second primer 251 isalso provided, and one or more additional cycles of denaturation, primerannealing, and extension are performed, e.g., by PCR, resulting indouble-stranded amplification product 215 (Panels E and F). In suchembodiments, product 215 typically corresponds to the amplified firstsurrogate nucleic acid to be detected. It will be evident that the pairof primers 250 and 251 define the 5′ ends of amplification product 215.It will also be evident that the amount of amplified first surrogatenucleic acid is proportional to the amount of target nucleic acidinitially present, the number of copies of the surrogate nucleic acidassociated with each copy of the target nucleic acid, and the number ofcycles of amplification performed.

As depicted in FIG. 2, the support capture probe includes a singlesequence U-2 and thus hybridizes to a single target capture probe.Optionally, however, a support capture probe can include two or moresequences U-2 and hybridize to two or more target capture probes.Similarly, as depicted, each of the target capture probes in the firstset includes an identical sequence U-1, and thus only a single supportcapture probe is needed; however, different target capture probes withina set optionally include different sequences U-1 and thus hybridize todifferent sequences U-2, within a single support capture probe ordifferent support capture probes on the surface of the support.

One or more blocking probes, single-stranded oligonucleotidescomplementary to region(s) of the target nucleic acid not occupied bythe target and surrogate capture probes, are optionally provided andhybridized to the target nucleic acid. The regions of the target nucleicacid to which the target capture probes, surrogate capture probes, andblocking probes hybridize can, but need not be, contiguous.

The methods can be used to detect target nucleic acids from essentiallyany type of sample. For example, the sample can be derived from ananimal, a human, a plant, a cultured cell, a virus, a bacterium, apathogen, and/or a microorganism. The sample optionally includes a celllysate, a tissue homogenate, an intercellular fluid, a bodily fluid(including, but not limited to, blood, serum, saliva, urine, sputum, orspinal fluid), and/or a conditioned culture medium, and is optionallyderived from a tissue (e.g., a formalin-fixed paraffin embedded tissue),a biopsy, and/or a tumor. Similarly, the target nucleic acid can beessentially any desired nucleic acid. As just a few examples, the firsttarget nucleic acid can be derived from one or more of an animal, ahuman, a plant, a cultured cell, a microorganism, a virus, a bacterium,or a pathogen. The first target nucleic acid can be essentially any typeof nucleic acid, e.g., a DNA, an RNA, an mRNA, a bacterial or viralgenomic RNA or DNA (double-stranded or single-stranded), a plasmid orother extra-genomic DNA, or another nucleic acid derived from amicroorganism (pathogenic or otherwise). The nucleic acid can bepurified, partially purified, or unpurified. It will be evident that atarget nucleic acid that is initially double-stranded will typically bedenatured before hybridization with target and/or surrogate captureprobes.

The methods can be conveniently multiplexed for detection of two or moretarget nucleic acids. Thus, in one aspect, the sample including thefirst target nucleic acid also includes a second target nucleic acid,and the methods include providing a second surrogate nucleic acid. Thesecond surrogate nucleic acid is physically associated with the secondtarget nucleic acid, to provide captured second surrogate nucleic acid.The captured second surrogate nucleic acid is amplified to provideamplified second surrogate nucleic acid, and the amplified secondsurrogate nucleic acid is detected. Presence or amount of the amplifiedsecond surrogate nucleic acid detected provides an indication ofpresence or amount of the second target nucleic acid in the sample.

The methods optionally include providing, capturing, and amplifyingthird, fourth, fifth, etc. surrogate nucleic acids as well, such thatfrom two to essentially any desired number of targets can be detectedsimultaneously. Essentially all of the features described for captureand amplification of the first surrogate nucleic acid above apply to theadditional surrogate nucleic acids as well. For example, typically asecond nucleic acid including the second surrogate nucleic acid and asecond sequence U-6, distinct from that attached to the first surrogatenucleic acid, is provided, and a second set of surrogate capture probescomplementary to the second target nucleic acid and to sequence(s)within U-6 is provided and used to associate the second surrogate andtarget nucleic acids. The second surrogate capture probes, secondsurrogate nucleic acid, and the like are preferably designed and/ortested to ensure minimal cross hybridization with the first surrogatenucleic acid, first target nucleic acid, and the like. It is worthnoting than m and n can, but need not be the same between first andsecond sets or subsets of target and/or surrogate capture probes.

A number of the detection methods described above can be multiplexed.For example, primers and/or probes labeled with different fluorescentlabels can be used to detect the amplified first and second surrogatenucleic acids. Typically, from two to four or five targets can beconveniently distinguished using fluorophores with distinct emissionspectra. Alternatively, as another example, essentially any number ofamplified surrogate nucleic acids can be physically separated and thendetected, permitting multiplex detection of even large numbers of targetnucleic acids simultaneously. Thus, in one class of embodiments,detecting the amplified first surrogate nucleic acid and the amplifiedsecond surrogate nucleic acid comprises physically separating theamplified first surrogate nucleic acid from the amplified secondsurrogate nucleic acid. Preferably, the amplified first and secondsurrogate nucleic acids contain different numbers of nucleotides, suchthat they can be conveniently separated based on their size. Separationcan be performed, for example, in a commercially available capillaryelectrophoresis system, such as the GenomeLab™ GeXP multiplexed PCRsystem from Beckman Coulter (on the world wide web at www (dot) beckman(dot) com).

As another alternative, the captured surrogate nucleic acids can beseparated from each other (e.g., by capturing the first and secondtarget nucleic acids and thus the first and second surrogate nucleicacids on different subsets of identifiable particles or at differentpositions on a spatially addressable solid support, for example) andthen amplified, prior to detection of the amplified surrogate nucleicacids.

The methods of the invention offer a number of advantages over currenttechniques for detecting and quantitating nucleic acids. For example,detection and quantitation of the target nucleic acid by capture andquantitation of a surrogate nucleic acid through quantitative real-timePCR (surrogate qPCR or sqPCR) has a number of advantages overtraditional quantitative PCR in which the target itself is amplified.Because surrogate qPCR measures the target nucleic acid indirectlythrough quantification of a surrogate nucleic acid (a surrogateamplicon), it avoids issues related to target amplification by qPCR suchas the necessity of primer/probe design and validation and assayoptimization for every target. Because essentially any target nucleicacid can be quantified through a common surrogate amplicon, thesurrogate nucleic acid, primers, and probes can be pre-designed andvalidated to possess close to 100% amplification efficiency and toensure no primer-dimer formation or mispriming events. In addition, themethods can be adapted to a variety of different qPCR platforms such asSYBR® Green, TaqMan®, molecular beacon, scorpion, Lux probe, Qzyme, andthe like.

Furthermore, use of surrogate nucleic acids avoids problems associatedwith design and validation of multiplex qPCR experiments. The surrogateqPCR methods described herein are well suited for multiplex analysis,because a common set of surrogate nucleic acids (surrogate amplicons),primers, and probes can be pre-designed and validated to ensure thatthey work together with close to 100% amplification efficiency and nomispriming or primer-dimer events. Once the standard set of amplicons isvalidated, essentially any combination of multiple nucleic acids can bedetected and quantified without the need for substantial upfront assaydesign and validation work-only the relevant set of target captureprobes, surrogate capture probes, and optional blocking probes need besynthesized for any new target nucleic acid. Only the probe sequencescomplementary to the desired target nucleic acid need be selected foreach new target, since the probe sequences complementary to the supportcapture probes and nucleic acids including the surrogate nucleic acidswould already have been designed and tested.

Surrogate qPCR is particularly well suited for quantifying mRNA becauseby quantifying a DNA surrogate nucleic acid, RNA isolation andconversion to cDNA by reverse transcription are avoided. The variationscaused by the pre-analytical steps is thus eliminated, and assayaccuracy and precision are therefore substantially improved. Additionalissues associated with RNA isolation and cDNA conversion, such asgenomic DNA contamination, 3′ bias, interference from other cDNAs withinthe nucleic acid mixture, and amplification efficiency variation amongdifferent samples, are also avoided as a result of surrogate ampliconquantification.

Surrogate qPCR is well suited for absolute quantification of the copynumber of a target nucleic acid in a sample by comparing PCR signal fromthe sample with PCR signal from a reference standard of surrogateamplicon. In surrogate qPCR, a standard curve is first constructed fromsurrogate amplicon of known concentrations. This curve is then used as areference standard for extrapolating quantitative information for targetnucleic acids of unknown concentrations. In standard qPCR assays, eitherRNA or cDNA standards are used for absolute quantification. Becausethese RNA or cDNA standards do not take into account the variationsassociated with RNA isolation and cDNA conversion in a real sample, theycannot give an accurate representation of the copy number of theintended target in the actual sample. In contrast, surrogate qPCRmeasures the same surrogate amplicon in reference standards as well asin a real sample; more accurate determination of copy number in thesample can thus be obtained.

Another advantage of surrogate qPCR is the “amplification” that isoptionally achieved during target capture and hybridization with thesurrogate amplicon. For example, in embodiments in which each copy ofthe target can bind to 10 or more copies of the surrogate amplicon, when10 copies of the target RNA are to be detected, 100 or more copies ofthe surrogate amplicon are actually being quantified. (As just a fewexamples of configurations in which 10 copies of a surrogate ampliconare bound to each copy of a target nucleic acid, 10 copies of a nucleicacid that includes one copy of the amplicon can be associated with thetarget, one copy of a nucleic acid that includes 10 copies of theamplicon can be associated with the target, five copies of a nucleicacid that includes two copies of the amplicon can be associated with thetarget, or two copies of a nucleic acid that includes five copies of theamplicon can be associated with the target.) This additionalamplification can minimize stochastic effects and improve assayprecision when measuring low copy number target nucleic acids.

Relative quantification can also be determined by surrogate qPCR withhigh precision and accuracy because the target and the reference controluse the same surrogate amplicon, so that the dynamic range as well asthe amplification efficiency of the target and reference is the same.

Background from nonspecific binding events can readily be determined forthe methods of the invention. For example, in surrogate qPCR,nonspecific binding of surrogate amplicon can optionally be determinedand subtracted from the specific capture of surrogate amplicon to itstarget. Two general types of nonspecific binding events are predicted topotentially occur: nonspecific binding of the surrogate nucleic acid tothe solid support, and undesirable probe-probe interactions. As shown inFIG. 3 Panel A, sample background is optionally determined by replacingtarget capture probes 305 with oligonucleotides 303 and 304, whichcorrespond to sequences U-1 and U-3, respectively, of the target captureprobes, so that target nucleic acid can not bind to the solid supportspecifically through target capture probe-support capture probeinteractions. (In addition to the oligonucleotides and the targetnucleic acid, Panel A depicts nucleic acid 311 including the surrogatenucleic acid, surrogate capture probes 307, support capture probes 302,and solid support 301.) In this situation, the only PCR signal leftresults from nonspecific binding, e.g., when the nucleic acid comprisingthe surrogate amplicon binds to the solid support nonspecifically.Because this assay background can be determined under exactly the sameassay conditions as the assay signal, it facilitates accuratedetermination of PCR signal contributed by the specific capture ofsurrogate amplicon to its target. In this situation, even theamplification efficiency and PCR inhibition can be controlled to thesame extent. Additional potential sources of background are shown inPanels B-D, probe background caused by probe-probe interactions thatresult in nonspecific capture of the surrogate nucleic acid to the solidsupport. Since probe background can be determined in the absence ofsample input, it can be known and minimized prior to assaying the targetnucleic acid in a sample. It is expected that probe background isgenerally low and can be negligible, particularly in embodiments inwhich two or more target capture probes and/or surrogate capture probesare required for stable capture of the target and/or surrogate nucleicacids. The background can determine the limit of detection of thesurrogate qPCR assay, and is therefore preferably minimized.

Surrogate qPCR can be applied to most, if not all, traditional qPCRapplications with higher assay accuracy and precision and greater easeof assay flow. For example, the methods of the invention can be appliedto quantitative gene expression analysis, microarray verification andfollow up, viral load determination, cancer diagnostics, pathogendetection, methylation detection, genotyping, GMO (genetically modifiedorganism) copy number determination, gene amplification and deletion,quantitative microsatellite analysis, prenatal diagnosis, single cellexpression analysis, and the like. Importantly, surrogate qPCR (andsimilar methods of the invention) provides an excellent platform formultiplex analysis in such applications.

Compositions

Compositions useful in practicing or produced by the methods herein formanother feature of the invention. Thus, one general class of embodimentsprovides a composition that includes a first target nucleic acid, anucleic acid comprising a first surrogate nucleic acid, a first set ofone or more surrogate capture probes, each of which is capable ofhybridizing simultaneously to the first target nucleic acid and to thenucleic acid comprising the first surrogate nucleic acid, and one ormore primers for amplifying the first surrogate nucleic acid.

The composition optionally also includes other reagents for amplifyingthe first surrogate nucleic acid, for example, a nucleic acidpolymerase, nucleoside or deoxynucleoside triphosphates, and the like.It also optionally includes amplified first surrogate nucleic acidand/or one or more reagents for detecting the amplified first surrogatenucleic acid. Exemplary detection reagents include, but are not limitedto, dyes, fluorescent primers, and fluorescent probes such as thosenoted above.

Essentially any desired number of copies of the surrogate nucleic acidcan be associated with each copy of the target nucleic acid. Thus, inone class of embodiments, the nucleic acid comprising the firstsurrogate nucleic acid is hybridized to the first set of surrogatecapture probes, which surrogate capture probes are hybridized to thefirst target nucleic acid, whereby the first surrogate nucleic acid isphysically associated with the first target nucleic acid at a molarratio of about 1:1. In another class of embodiments, the nucleic acidcomprising the first surrogate nucleic acid is hybridized to the firstset of surrogate capture probes, which surrogate capture probes arehybridized to the first target nucleic acid, whereby the first surrogatenucleic acid is physically associated with the first target nucleic acidat a molar ratio of at least about 2:1, at least about 3:1, at leastabout 5:1, or at least about 10:1 first surrogate nucleic acid:firsttarget nucleic acid. As in the methods described above, the nucleic acidcomprising the surrogate nucleic acid can include one copy of thesurrogate nucleic acid, or it can include two or more copies of thesurrogate nucleic acid. Thus, in embodiments in which multiple copies ofthe surrogate nucleic acid are associated with or captured to each copyof the target nucleic acid, more than one copy of the nucleic acidcomprising the surrogate nucleic acid can be associated with the eachcopy of the target nucleic acid and/or each copy of the nucleic acidcomprising the surrogate nucleic acid can include more than one copy ofthe surrogate nucleic acid.

In one class of embodiments, the composition includes a solid support towhich the target and surrogate nucleic acids can be (or are) captured,for example, a multiwell plate or a plurality of particles. Exemplarysupports are provided in the section entitled “Solid supports” below.

Essentially all of the features described for the methods above apply tothese embodiments as well, as relevant, for example, with respect tonumber and configuration of surrogate capture probes, number andconfiguration of target capture probes, support capture probes, types oftarget and surrogate nucleic acids, optional blocking probes, and/or thelike.

It is worth noting that the composition optionally also includes asecond nucleic acid comprising a second surrogate nucleic acid, a secondset of one or more surrogate capture probes, each of which surrogatecapture probes is capable of hybridizing simultaneously to a secondtarget nucleic acid and to the second nucleic acid comprising the secondsurrogate nucleic acid, the second target nucleic acid, one or moreprimers for amplifying the second surrogate nucleic acid, amplifiedsecond surrogate nucleic acid, and/or one or more reagents for detectingthe amplified second surrogate nucleic acid. Third, fourth, fifth, etc.target and surrogate nucleic acids, sets of surrogate capture probes,and the like are optionally also present in the composition.

Kits

Yet another general class of embodiments provides a kit for detecting atleast one target nucleic acid. The kit includes a nucleic acidcomprising a first surrogate nucleic acid, a first set of one or moresurrogate capture probes, each of which is capable of hybridizingsimultaneously to a first target nucleic acid and to the nucleic acidcomprising the first surrogate nucleic acid, a solid support comprisinga first support capture probe bound to the solid support, and a firstset of m target capture probes, where m is at least one, which targetcapture probes are capable of hybridizing simultaneously to the firsttarget nucleic acid and to the first support capture probe, packaged inone or more containers.

The kit optionally also includes one or more primers for amplifying thefirst surrogate nucleic acid, other reagents for amplifying the firstsurrogate nucleic acid (e.g., a nucleic acid polymerase, nucleoside ordeoxynucleoside triphosphates, and the like), one or more reagents fordetecting an amplified first surrogate nucleic acid (e.g., a dye or afluorescently labeled primer or probe), a wash buffer for removingmaterials not specifically captured on the solid support, a lysis bufferfor lysing cells and/or homogenizing tissues, the target and/or thesurrogate nucleic acid at a standard concentration, and/or instructionsfor using the kit to detect and optionally quantitate one or morenucleic acids.

Essentially all of the features described for the methods andcompositions above apply to these embodiments as well, as relevant, forexample, with respect to the number of copies of the surrogate nucleicacid associated with each copy of the target nucleic acid, number andconfiguration of surrogate capture probes, number and configuration oftarget capture probes, support capture probes, types of target andsurrogate nucleic acids, type of solid support, optional blockingprobes, and/or the like.

It is worth noting that the kit optionally also includes a secondnucleic acid comprising a second surrogate nucleic acid, a second set ofone or more surrogate capture probes, each of which surrogate captureprobes is capable of hybridizing simultaneously to a second targetnucleic acid and to the second nucleic acid comprising the secondsurrogate nucleic acid, and a second set of m target capture probes,where m is at least one, which target capture probes are capable ofhybridizing simultaneously to the second target nucleic acid and to thefirst support capture probe (or, alternatively, to a second supportcapture probe bound to the solid support). One or more primers foramplifying the second surrogate nucleic acid and/or one or more reagentsfor detecting the amplified second surrogate nucleic acid can also beincluded in the kit, as can third, fourth, fifth, etc. target andsurrogate nucleic acids, sets of target and surrogate capture probes,and the like.

Systems

In one aspect, the invention includes systems, e.g., systems used topractice the methods herein and/or comprising the compositions describedherein. The system can include, e.g., a fluid and/or particle handlingelement, a fluid and/or particle containing element, a laser forexciting a fluorescent label or labels, a detector for detectingfluorescent emissions from a fluorescent label or labels, a thermalcycler, a capillary electrophoresis instrument, and/or a robotic elementthat moves other components of the system from place to place as needed(e.g., a multiwell plate handling element). For example, in one class ofembodiments, a composition of the invention is contained in a thermalcycler, a microplate reader, or like instrument. In one class ofembodiments, the system automates capture, amplification, and/ordetection of one or more target nucleic acids.

The system can optionally include a computer. The computer can includeappropriate software for receiving user instructions, either in the formof user input into a set of parameter fields, e.g., in a GUI, or in theform of preprogrammed instructions, e.g., preprogrammed for a variety ofdifferent specific operations. The software optionally converts theseinstructions to appropriate language for controlling the operation ofcomponents of the system (e.g., for controlling a fluid handlingelement, thermal cycler, robotic element, and/or laser). The computercan also receive data from other components of the system, e.g., from adetector, and can interpret the data, provide it to a user in a humanreadable format, or use that data to initiate further operations, inaccordance with any programming by the user.

Solid Supports

Certain embodiments described herein employ a solid support. The solidsupport can be essentially any suitable support, including any of avariety of materials, configurations, and the like. For example, in oneclass of embodiments, the solid support is a substantially planar solidsupport, e.g., an upper surface of the bottom of a well of a multiwellplate, a slide, a membrane, or the like. Similarly, suitable solidsupports include any surface of a well of a multiwell plate, whetherplanar or not. As another example, the solid support can comprise aplurality of particles, e.g., microspheres, beads, cylindricalparticles, irregularly shaped particles, or the like. Optionally, theparticles (or other solid support) are functionalized, for example, forease of attachment of support or target capture probes. The particlesoptionally have additional or other desirable characteristics. Forexample, the particles can be magnetic or paramagnetic, providing aconvenient means for separating the particles from solution, e.g., tosimplify separation of the particles from any materials not bound to theparticles. Similarly, for example, individual particles or sets ofparticles are optionally identifiable, e.g., by an embedded fluorescentor other code. The solid support is optionally spatially addressable.

A variety of suitable solid supports are commercially readily available.For example, microspheres with a variety of surface chemistries arecommercially available, e.g., from Dynal (on the world wide web at www(dot) dynalbiotech (dot) com; microspheres available includecarboxylated magnetic Dynabeads® with surface streptavidin),Polysciences, Inc. (on the world wide web at www (dot) polysciences(dot) com) or Luminex Corporation (on the world wide web at www (dot)luminexcorp (dot) com; microspheres available include fluorescentlycoded, identifiable sets of microspheres). For example, microsphereswith carboxyl or amino groups are available and permit covalent couplingof molecules (e.g., polynucleotide probes with free reactive groups) tothe microspheres. As another example, microspheres with surfacestreptavidin are available and can bind biotinylated capture probes;similarly, microspheres coated with biotin are available for bindingcapture probes conjugated to avidin or streptavidin. As another example,surface-modified and pre-coated slides with a variety of surfacechemistries are commercially available, e.g., from TeleChemInternational (on the world wide web at www (dot) arrayit (dot) com),Corning, Inc. (Corning, N.Y.), or Greiner Bio-One, Inc. (on the worldwide web at www (dot) greinerbiooneinc (dot) com). For example,silanated and silyated slides with free amino and aldehyde groups,respectively, are available and permit covalent coupling of molecules(e.g., polynucleotides with free aldehyde, amine, or other reactivegroups) to the slides. As another example, slides with surfacestreptavidin are available and can bind biotinylated capture probes. Asyet another example, surface-modified and pre-coated multiwell platesare commercially available, e.g., from Sigma-Aldrich, Inc. (on the worldwide web at www (dot) sigmaaldrich (dot) com). For example, streptavidinand poly-D-lysine coated multiwell plates are available.

Protocols for using such commercially available microspheres, plates,and slides (e.g., methods of covalently coupling polynucleotides tocarboxylated microspheres for use as capture probes, methods of blockingreactive sites on the support surface that are not occupied by thepolynucleotides, methods of binding biotinylated polynucleotides toavidin-functionalized supports, and the like) are typically suppliedwith the supports and are readily utilized and/or adapted by one ofskill. In addition, coupling of reagents to microspheres is welldescribed in the literature. For example, see Yang et al. (2001) “BADGE,Beads Array for the Detection of Gene Expression, a high-throughputdiagnostic bioassay” Genome Res. 11:1888-98; Fulton et al. (1997)“Advanced multiplexed analysis with the FlowMetrix™ system” ClinicalChemistry 43:1749-1756; Kellar and Iannone (2002) “Multiplexedmicrosphere-based flow cytometric assays” Experimental Hematology30:1227-1237; U.S. Pat. No. 5,981,180 entitled “Multiplexed analysis ofclinical specimens apparatus and methods” to Chandler et al. (Nov. 9,1999); U.S. Pat. No. 6,449,562 entitled “Multiplexed analysis ofclinical specimens apparatus and methods” to Chandler et al. (Sep. 10,2002); and references therein.

Molecular Biological Techniques

In practicing the present invention, many conventional techniques inmolecular biology, microbiology, and recombinant DNA technology areoptionally used. These techniques are well known and are explained in,for example, Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego,Calif.; Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000; and Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2006). Other useful references, e.g. for cell isolation and culture(e.g., for subsequent nucleic acid or protein isolation) includeFreshney (1994) Culture of Animal Cells, a Manual of Basic Technique,third edition, Wiley-Liss, New York and the references cited therein;Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems JohnWiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (Eds.) (1995)Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer LabManual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks(Eds.) The Handbook of Microbiological Media (1993) CRC Press, BocaRaton, Fla.

Making Polynucleotides

Methods of making nucleic acids (e.g., by in vitro amplification,purification from cells, or chemical synthesis), methods formanipulating nucleic acids (e.g., by restriction enzyme digestion,ligation, etc.), and various vectors, cell lines and the like useful inmanipulating and making nucleic acids are described in the abovereferences. In addition, essentially any polynucleotide (including,e.g., labeled or biotinylated polynucleotides) can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company (on the world wide web at www (dot) mcrc (dot)com), The Great American Gene Company (on the world wide web at www(dot) genco (dot) com), ExpressGen Inc. (on the world wide web at www(dot) expressgen (dot) com), Qiagen (on the internet at oligos (dot)qiagen (dot) com) and many others.

A label, biotin, or other moiety can optionally be introduced to apolynucleotide, either during or after synthesis. For example, a biotinphosphoramidite can be incorporated during chemical synthesis of apolynucleotide. Alternatively, any nucleic acid can be biotinylatedusing techniques known in the art; suitable reagents are commerciallyavailable, e.g., from Pierce Biotechnology (on the world wide web at www(dot) piercenet (dot) com). Similarly, any nucleic acid can befluorescently labeled, for example, by using commercially available kitssuch as those from Molecular Probes/Invitrogen (at probes (dot)invitrogen (dot) com/) or Pierce Biotechnology (on the world wide web atwww (dot) piercenet (dot) com) or by incorporating a fluorescentlylabeled phosphoramidite during chemical synthesis of a polynucleotide.

Specialized primers and probes can also be custom or standard orderedfrom any of a variety of commercial sources. For example, custom TaqMan®primers can be ordered from Applied Biosystems (on the world wide web atwww (dot) appliedbiosystems (dot) com), molecular beacons fromIntegrated DNA Technologies or The Midland Certified Reagent Company (onthe world wide web at www (dot) idtdna (dot) com and www (dot) oligos(dot) com, respectively), Invader® oligonucleotides from Third WaveTechnologies, Inc. (on the world wide web at www (dot) twt (dot) com),and scorpion primers from DxS (on the world wide web at www (dot)dxsgenotyping (dot) com), as just a few examples.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. Accordingly, the following examples areoffered to illustrate, but not to limit, the claimed invention.

Example 1 Detection of a Nucleic Acid Target Using Surrogate QPCR

The following sets forth a series of experiments that demonstratedetection of an IL-6 RNA target by capturing a surrogate nucleic acid tothe IL-6 target and then amplifying the captured surrogate nucleic acidand detecting the amplified surrogate nucleic acid, using quantitativereal-time PCR.

Materials

The sequence of the nucleic acid comprising the surrogate nucleic acidwas 5′CGGGTATGGCTTTCATGTGGTTCTGGACAATGACGGTTACGGAGGTGGGCGTGGTCGTCTGCTGGGTTGGTCACGTGGGCGATCGACTTTTTAAAACGGTAACTTCA TGCTTTGACTCAG(SEQ ID NO:1); 5T separate the surrogate nucleic acid (the surrogateamplicon, underlined) and the sequence U-6 that binds to the surrogatecapture probe. Primers used for qPCR amplification of the surrogateamplicon were as follows: forward primer 5′ CGGGTATGGCTTTCATGTGGT (SEQID NO:2) and reverse primer 5′ gtcgatcgcccacgtgac (SEQ ID NO:3). Invitro transcribed (IVT) IL-6 RNA was used as the target nucleic acid, inthe indicated amounts. The sequence of the support capture probe was 5′TTTTTTactttctttccaagag (SEQ ID NO:4). Sequences of the target captureprobes, surrogate capture probes, and blocking probes are listed inTable 1.

TABLE 1Sequences of IL-6 probe set: target capture probes (TCP), surrogatecapture probes (SCP), and blocking probes (BP). SEQ ID NO: 5 SCPaagaggtgagtggctgtctgtgTTTTTctgagtcaaagcatgaagttacc gtttt SEQ ID NO: 6SCP gaatttgtttgtcaattcgttctgTTTTTctgagtcaaagcatgaagtta ccgttttSEQ ID NO: 7 SCP atctgttctggaggtactctaggtataTTTTTctgagtcaaagcatgaagttaccgtttt SEQ ID NO: 8 SCPggcttgttcctcactactctcaaTTTTTctgagtcaaagcatgaagttac cgtttt SEQ ID NO: 9SCP ctgcaggaactggatcaggacTTTTTctgagtcaaagcatgaagttaccg ttttSEQ ID NO: 10 SCP gcatctagattctttgcctttttTTTTTctgagtcaaagcatgaagttaccgtttt SEQ ID NO: 11 SCPtgtgcctgcagcttcgtcaTTTTTctgagtcaaagcatgaagttaccgtt tt SEQ ID NO: 12 SCPtgtcctgcagccactggttcTTTTTctgagtcaaagcatgaagttaccgt ttt SEQ ID NO: 13 SCPggtttctgaccagaagaaggaatgTTTTTctgagtcaaagcatgaagtta ccgtttt SEQ ID NO: 14SCP aagttctgtgcccagtggacaTTTTTctgagtcaaagcatgaagttaccg ttttSEQ ID NO: 15 BP TGGGGCAGGGAAGGCA SEQ ID NO: 16 BP GGAATCTTCTCCTGGGGGTACSEQ ID NO: 17 BP TGGGGCGGCTACATCTTT SEQ ID NO: 18 BPGCTTTCACACATGTTACTCTTGTTACA SEQ ID NO: 19 BP TTTGGAAGGTTCAGGTTGTTTTSEQ ID NO: 20 BP CCTCAAACTCCAAAAGACCAGTG SEQ ID NO: 21 BPTTGGGTCAGGGGTGGTTATT SEQ ID NO: 22 BP CTGCAGGAACTCCTTAAAGCTGSEQ ID NO: 23 BP CCCATTAACAACAACAATCTGAGG SEQ ID NO: 24 BPggctcctggaggcgagata SEQ ID NO: 25 BP aactggaccgaaggcgct SEQ ID NO: 26 BPgcaggcaacaccaggagc SEQ ID NO: 27 BP gatgccgtcgaggatgtacc SEQ ID NO: 28BP ctgccagtgcctctttgct SEQ ID NO: 29 BP gcatccatctttttcagccatcSEQ ID NO: 30 BP atgattttcaccaggcaagtct SEQ ID NO: 31 BPttttgtactcatctgcacagctct SEQ ID NO: 32 BP gcaggctggcatttgtggSEQ ID NO: 33 BP cgcagaatgagatgagttgtca SEQ ID NO: 34 BPtgcccatgctacatttgcc SEQ ID NO: 35 TCPgagcttctctttcgttcccgTTTTTctcttggaaagaaagt SEQ ID NO: 36 TCPtgtggagaaggagttcatagctgTTTTTctcttggaaagaaagt SEQ ID NO: 37 TCPagccccagggagaaggcTTTTTctcttggaaagaaagt SEQ ID NO: 38 TCPtgtctcctttctcagggctgaTTTTTctcttggaaagaaagt SEQ ID NO: 39 TCPcctcattgaatccagattggaaTTTTTctcttggaaagaaagt SEQ ID NO: 40 TCPgaagagccctcaggctggaTTTTTctcttggaaagaaagt

To conjugate magnetic beads with support capture probe, Dynal Dynabeads®M270 carboxylic acid beads were conjugated with 5′ amine labeled supportcapture probe according to Flagella et al. (2006) “A multiplex branchedDNA assay for parallel quantitative gene expression profiling” AnalBiochem. 352:50-60. Briefly, 2.5×10⁸ beads were resuspended in 200 μl0.1 M MES, pH 4.5, and incubated in the presence of ˜8 μM supportcapture probe and 2 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (Pierce, Rockford, Ill.).

Surrogate qPCR

An initial experiment was performed to determine whether surrogatenucleic acids captured to target nucleic acids bound to magnetic beadsolid supports could be amplified in the presence of the beads orwhether the surrogate nucleic acid would need to be isolated from thebeads prior to amplification. Surrogate nucleic acid was amplified bySYBR® green qPCR in the presence (squares) or absence (diamonds) ofapproximately one million magnetic beads, and, as shown in FIG. 4 PanelA, the presence of magnetic beads in qPCR reactions had minimal effect.In subsequent experiments, amplification of surrogate nucleic acidcaptured to beads was therefore performed in the presence of the beads.

Samples containing IL-6 IVT RNA were mixed with the pooled probe set(the target capture probes, surrogate capture probes, and blockingprobes) and capture beads (support capture probe-conjugated magneticbeads, 300,000 beads per assay) and hybridized for 16 hours at 53° C. in100 μL volume. The components in a 100 μl IVT RNA assay were 33% v/vlysis mixture, 40% v/v capture buffer, 1 μg tRNA, and the panel-specificprobe set (target capture probe, 0.165 fmol/μl; surrogate capture probe,0.66 fmol/μl; blocking probe, 0.33 fmol/μl). Lysis mixture and capturebuffer commercially available from Panomics, Inc. (www (dot) panomics(dot) com), e.g., as catalog numbers QG0502 and QG0518, respectively,were used in these experiments, but it will be evident that any of avariety of similar suitable solutions can be employed (for example, thecapture diluent described in Collins et al. (1997) Nucleic Acid Research25:2979-2984 (127 mM LiCl, 5% lithium lauroyl sulfate, 9 mM EDTA, 50 mMHEPES (pH 7.5), 0.05% hespan (DuPont Pharmaceuticals), 0.05% ProClin 300(Supelco), 0.2% casein (Research Organics, Hammarsten quality) isoptionally employed as the lysis mixture and/or 50 mM HEPES acid, 50 mMHEPES sodium salt, 1% lithium lauryl sulfate, 8 mM EDTA, 0.3% nucleicacid blocking agent (Roche), and 0.5% Micro-O-protect is optionallyemployed as the capture buffer).

After the overnight hybridization step, unbound materials were washedfrom the beads (complexed with the probe set and IL-6 target RNA) usinga magnetic separator and addition of wash buffer (0.1×SSC, 0.03% lithiumlauryl sulfate). Five washes were performed. The beads were thenincubated with 100 fmol of the nucleic acid molecule including thesurrogate nucleic acid in amplifier/label probe diluent (Panomicscatalog number QG0505; 3M tetramethyl ammonium chloride, 0.1% Sarkosyl,50 mM Tris-HCl, 4 mM EDTA, 4% dextran sulfate, 1% BSA and 0.5% v/vMicr-O-protect (Roche Molecular Systems, Pleasanton, Calif.) isoptionally employed instead) for 1 hour at 46° C. After thehybridization with surrogate nucleic acid, the beads were further washedfive times with the wash buffer and then transferred to qPCR plate forSYBR® qPCR. 25 uL per well of SYBR® qPCR reaction was set up usingTakara's SYBR® qPCR kit. For control reactions, serial dilutions of thenucleic acid molecule including the surrogate nucleic acid were rundirectly in SYBR® qPCR.

PCR reaction mixture contained:

Final Reagents Volume Concentration SYBR ® Premix Ex Taq ™ 12.5 uL 1xPCR forward primer (10 uM) 0.5 uL 0.2 uM PCR reverse primer (10 uM) 0.5uL 0.2 uM ROX ™ Reference Dye II (50x) 0.5 uL 1x Bead-associatedsurrogate 2 uL amplicon dH2O 9 uL Total 25 uL.

PCR conditions using Stratagene's Mx4000® qPCR system were:

Segment 1: Initial denaturation

Repetitions: 1

95° C. 10 sec

Segment 2: PCR

Repetitions: 40

95° C. 5 sec

60° C. 20 sec

Results of the sqPCR, in which the surrogate nucleic acid was capturedto the IL-6 target and then amplified and detected, are shown in FIG. 4Panel B (diamonds). Results from control amplifications of the surrogatenucleic acid (i.e., dilutions of the surrogate nucleic acid, notcaptured to the target) are also shown (squares). The surrogate qPCRexperiment successfully detected the IL-6 target and permitted itsquantitation even at amounts as low as 0.004 amol (2500 molecules).

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1-26. (canceled)
 27. A composition comprising: a first target nucleicacid; a nucleic acid comprising a first surrogate nucleic acid; a firstset of one or more surrogate capture probes, each of which surrogatecapture probes is capable of hybridizing simultaneously to the firsttarget nucleic acid and to the nucleic acid comprising the firstsurrogate nucleic acid; and one or more primers for amplifying the firstsurrogate nucleic acid.
 28. The composition of claim 27, wherein thenucleic acid comprising the first surrogate nucleic acid is hybridizedto the first set of surrogate capture probes, which surrogate captureprobes are hybridized to the first target nucleic acid, whereby thefirst surrogate nucleic acid is physically associated with the firsttarget nucleic acid at a molar ratio of about 1:1.
 29. The compositionof claim 27, wherein the nucleic acid comprising the first surrogatenucleic acid is hybridized to the first set of surrogate capture probes,which surrogate capture probes are hybridized to the first targetnucleic acid, whereby the first surrogate nucleic acid is physicallyassociated with the first target nucleic acid at a molar ratio of atleast about 2:1, at least about 3:1, at least about 5:1, or at leastabout 10:1 first surrogate nucleic acid:first target nucleic acid. 30.The composition of claim 27, wherein one or more copies of the nucleicacid comprising the first surrogate nucleic acid are associated with acopy of the first target nucleic acid; wherein the first set ofsurrogate capture probes comprises a subset of surrogate capture probesfor each of the one or more copies of the nucleic acid comprising thefirst surrogate nucleic acid; and wherein each subset of surrogatecapture probes comprises n surrogate capture probes, where n is at leasttwo.
 31. The composition of claim 27, wherein the first set of surrogatecapture probes comprises two or more surrogate capture probes.
 32. Thecomposition of claim 31, wherein the surrogate capture probes hybridizeto nonoverlapping polynucleotide sequences in the first target nucleicacid.
 33. The composition of claim 27, comprising a solid support. 34.The composition of claim 33, wherein the solid support comprises aplurality of particles.
 35. The composition of claim 33, comprising afirst set of m target capture probes, where m is at least one, whichtarget capture probes are capable of hybridizing to the first targetnucleic acid.
 36. The composition of claim 35, wherein the targetcapture probes hybridize to nonoverlapping polynucleotide sequences inthe first target nucleic acid.
 37. The composition of claim 35, whereinm is at least two.
 38. The composition of claim 35, wherein the solidsupport comprises a first support capture probe bound to the solidsupport, and wherein the target capture probes of the first set arecapable of hybridizing simultaneously to the first target nucleic acidand to the first support capture probe.
 39. The composition of claim 27,comprising amplified first surrogate nucleic acid.
 40. The compositionof claim 39, comprising one or more reagents for detecting the amplifiedfirst surrogate nucleic acid.
 41. (canceled)
 42. The composition ofclaim 27, wherein the first surrogate nucleic acid is a DNA. 43-44.(canceled)
 45. The composition of claim 27, comprising a second nucleicacid comprising a second surrogate nucleic acid; a second set of one ormore surrogate capture probes, each of which surrogate capture probes iscapable of hybridizing simultaneously to a second target nucleic acidand to the second nucleic acid comprising the second surrogate nucleicacid; and the second target nucleic acid and/or one or more primers foramplifying the second surrogate nucleic acid.
 46. A kit comprising: anucleic acid comprising a first surrogate nucleic acid; a first set ofone or more surrogate capture probes, each of which surrogate captureprobes is capable of hybridizing simultaneously to a first targetnucleic acid and to the nucleic acid comprising the first surrogatenucleic acid; a solid support comprising a first support capture probebound to the solid support; a first set of m target capture probes,where m is at least one, which target capture probes are capable ofhybridizing simultaneously to the first target nucleic acid and to thefirst support capture probe; and one or more primers for amplifying thefirst surrogate nucleic acid; packaged in one or more containers. 47-48.(canceled)
 49. The kit of claim 46, further comprising: a second nucleicacid comprising a second surrogate nucleic acid; a second set of one ormore surrogate capture probes, each of which surrogate capture probes iscapable of hybridizing simultaneously to a second target nucleic acidand to the second nucleic acid comprising the second surrogate nucleicacid; and a second set of m target capture probes, where m is at leastone, which target capture probes are capable of hybridizingsimultaneously to the second target nucleic acid and to the firstsupport capture probe.
 50. The composition of claim 30, wherein the 5′end of each of the surrogate capture probes in the first set hybridizesto the first target nucleic acid while the 3′ end hybridizes to thenucleic acid comprising the first surrogate nucleic acid, or wherein the3′ end of each of the surrogate capture probes in the first sethybridizes to the first target nucleic acid while the 5′ end hybridizesto the nucleic acid comprising the first surrogate nucleic acid.
 51. Thecomposition of claim 27, wherein the first surrogate nucleic acid isphysically associated with the first target nucleic acid.